New Materials For The Construction of Electrochemical Bosensors

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Chapter 1
New Materials for the Construction of Electrochemical

Biosensors
Robert Sndulescu, Mihaela Terti,

Cecilia Cristea and Ede Bodoki
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/60510
Abstract
The development of electrochemical sensors has attracted great interest due to these
sensors high sensitivity and selectivity. Here, we present the general concept and the
classification of biosensors, their advantages and drawbacks, the main strategies in
electrochemical biosensor technology and the materials used in electrochemical
sensors, such as electrodes and supporting substrates, materials for improved
sensitivity and selectivity, materials for bioreceptor immobilization, and biological
recognition elements. Various nanomaterials, such as carbon-based materials (carbon
nanotubes, graphene, carbon nanoparticles), inorganic and organic nanoparticles
(magnetic and metal nanoparticles, nanosized clays), conductive and insulating
polymers (nanosized and nanostructured polymers, molecularly imprinted poly
mers), and hybrid materials, etc., have been successfully applied for the enhancement
of the electroanalytical performance of biosensors and for the immobilization of
biorecognition elements. Among these, due to their unique physiochemical features,
carbon-based materials, such as carbon nanotubes and graphenes, have received
special attention in recent years, and examples of surface functionalization using
various types of nanoparticles are presented. The future trends in sensor research
activities and areas of development that are expected to have an impact in biosensor
performance, like immobilization techniques, nanotechnology, miniaturization and
multisensor array determinations, are also examined.
Keywords: Carbon-based nanomaterials, metal nanoparticles, magnetic nanoparticles, nanostruc

ture, molecularly imprinted polymers
2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.
2 Biosensors - Micro and Nanoscale Applications
1. Introduction
The development of electrochemical sensors has attracted a great deal of interest due to their
high sensitivity and selectivity, and they are being increasingly used in many fields, such as
analytical chemistry, industrial process monitoring and control, clinical diagnostics, environ
mental monitoring and security, and food safety. By adding an enzyme at the electrode surface,
they can also gain high specificity: thus the biosensor research area was born.
Biosensors (analytical devices coupling a transducer with a biorecognition element) are
attractive for pharmaceutical and biomedical analysis due to their sensitivity (ng/mL or less)
and high selectivity, and sometimes their specificity, high benefit/cost ratio, simple use and
rapidity of data collection [1].
The major advantages of biosensors over traditional analytical methods, which will certainly
lead in the near future to their even more pronounced use in the biomedical field, are: the fact
that analyte detection can very often be made without prior separation; the short response
times that make possible the real-time monitoring of biological and manufacturing processes;
their ease of use, allowing in-field or point-of-care measurements; the flexibility and simplicity
of preparation; the possibility of mass production and low production costs; and the possibility
of miniaturization and automatization. Miniaturization is of great importance because
biological samples are available in small amounts, and tissue damage must be minimized in
cases of in-vivo monitoring. Therefore, the use of biosensors as components of modern medical
devices has improved their portability, functionality and reliability for point-of-care analysis
and real-time diagnosis [2].
However, many amperometric biosensors described in the recent literature still display a few
drawbacks compared to other analytical methods. The most difficult problems to overcome
for biosensors with biomedical applications are: the reduced stability, the electrochemical
interferences, and the lack of or low response reproducibility. Removed from their natural
environment, most biocomponents tend to rapidly lose their activity and thus limit the lifetime
of the sensor. Modifying electrode surfaces in a way that exclusively favours one single
electrochemical process is a difficult task and sample matrices in the biomedical field are very
complex. Last but not least, in the case of in-vivo measurements biocompatibility and biofoul
ing are critical issues [3].
The selectivity of the biosensor for the target analyte is mainly determined by the biorecogni
tion element, whilst the sensitivity of the biosensor is greatly influenced by the transducer.
New sensing elements like genetically transformed cells and genetically engineered receptor
molecules can improve affinity and specificity; therefore, genetic engineering and mass
production of the molecular recognition may ultimately dictate the success of biosensor
technologies. Enzyme variants that are specific for individual analytes have already been
obtained by genetic modification, and novel gene fusions will lead to more sensitive
biosensors [4].
As for the electrochemical transducer, important advances have been recently made thanks
to the introduction of new platforms for biosensor design, such as nanotechnological
New Materials for the Construction of Electrochemical Biosensors 3
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materials and nanostructured architectures (i.e., nanoparticles, carbon nanotubes and

nanofibres, graphenes, nanostructured surfaces, etc.), which have improved the sensitivity
of the assembly [5].
2. Strategies of electrochemical biosensor technology
The biosensor development and construction strategy includes five features: 1) the detected
or measured parameter and the matrix, 2) the working principle of the transducer, 3) the
chemical/biochemical model, 4) the field of application and 5) the technology and materials
for sensor fabrication [6].
Materials generally used for electrochemical sensors are classified as: (1) materials for the
electrode and supporting substrate, (2) materials for improving electroanalytical performan
ces, (3) materials for the immobilization of biological recognition elements and (4) biological
elements; the last two are applicable for electrochemical biosensors [5].
Materials used for the electrode and supporting substrate are usually conductive materials
exhibiting low currents in an electrolyte solution, free of any electroactive species, over a
relatively wide potential window. Among the most frequently used materials for the electrode
and supporting substrate, the following may be mentioned: metals (mercury, platinum, gold,
silver and stainless steel), metal oxides (indium tin oxide, ITO) carbon-based materials (glassy
carbon, graphite, carbon black and carbon fibre), new hybrid materials, and organic electro
conductive polymers or salts. Boron-doped diamond is a special case of material, because bare
diamond is a non-conducting, allotropic form of carbon. In the last decade, in order to avoid
mercury-based electrodes (which are highly toxic), different metallic films (Au, Hg, Bi, Ag, Pb,
Sn, Sb, Co, Ga, Se) that are relatively simple to achieve and easy to reproduce were developed.
Some strategies to develop two- and three-dimensional nanostructured electrodes with larger
surfaces (mesoporous silicates, metal oxides, polymers or carbons) were also recently reported
[7]. The major challenge in the field of electrochemical sensors, consisting in the improvement
of their electroanalytical performances, mainly in terms of sensitivity and selectivity, was
addressed by the development of nanotechnology (especially nanoparticles, carbon nanotubes
and graphenes) and nanostructured architectures, and this will be detailed in section 3.
The stable immobilization of a bioelement on an electrode surface, with complete reten
tion of its biological activity and good diffusion properties for substrates, is a crucial
problem for the commercial development of biosensors. A successful matrix should stably
immobilize or integrate biomolecules at the transducers surface, it should efficiently
maintain their functionality, while providing accessibility for the target analyte, and it
should also ensure an intimate contact with the transducer surface [8]. The immobiliza
tion matrix may function purely as a support or may also be involved in the mechanism
of signal transduction mediation. The selection of an appropriate immobilization method
depends on the nature of the biological element, the type of transducer used, the physico
chemical properties of the analyte and the biosensors operating conditions [9]. The choice
of materials for the immobilization of biological recognition elements strongly depends on
the employed procedure. The procedures that may be employed are as follows: 1) Entrap
ment behind a permeable membrane as a thin film covering the electrochemical detector
(dialysis membrane) [10]; 2) Entrapment within a polymeric matrix (polyacrylonitrile, agar
gel, polyurethane or poly(vinyl-alcohol) membranes, sol gels or redox hydrogels with redox
centres such as [Os(bpy)2Cl]+, conducting polymers [11]; 3) Entrapment within self-assem
bled monolayers (SAMs) [12] or bilayer lipid membranes (BLMs) [13]; 4) Covalent bond
ing on membranes or surfaces activated by means of bifunctional groups or spacers
(glutaraldehyde, carbodiimide, SAMs or multilayers, avidin-biotin, silanization) [14]; 5) Bulk
modification of the electrode material (graphite epoxy resin or carbon paste modified with
polyoxometallates, clays and double layered hydroxides, zeolites, functionalized silica, sol-
gel-derived inorganic and hybrid materials, nanomaterials, sparingly soluble or insoluble
inorganic salts, molecular and macrocyclic ligands, complex compounds, organic poly
mers, surfactants and lipids) [7].
The biorecognition elements used for biosensor development are classified into biologi
cal and artificial (biomimetic) receptors. They are selected depending on the application
and the desired performance criteria, based on the following interaction principles: enzyme/
substrate, antigen/antibody, mRNA/DNA, PNA/DNA or RNA, microorganism/substrate or
toxic molecules, cell receptor/hormone and (semi)synthetic receptor/target molecule [15].
Initially, three types of biorecognition elements were identified [16]: 1) Enzyme (mono or
multienzyme), the most common and best developed recognition system, including
antibodies, natural receptors and nucleic acids; 2) Whole cells (microorganisms, such as
bacteria, fungi, eukaryotic cells or yeast), or cell organelles or particles (mitochondria, cell
walls); 3) Tissue (plant or animal tissue slice). More recently, the range of biochemical
receptors was extended with biomimetic recognition elements (aptamers, ribosymes and
molecularly imprinted polymers) [17]. The biological recognition elements are based either
on catalytic or bioaffinity features, whilst biomimetic ones are based on biocomplexing and
bioaffinity.
The biocatalytic recognition sensors are based on a reaction catalysed by macromolecules,

either in their original biological environment, in their isolated form, or as a product of
semisynthesis. Thus, a continuous consumption of substrate(s) is achieved by the immobilized
biocatalyst incorporated into the sensor, and transient or steady-state responses are monitored
by the integrated detector. In the construction of biosensors, enzymes are the most commonly
used biological elements, despite their rather high extraction, isolation and purification costs,
as they rapidly and cleanly form selective bonds with the substrate. Among the enzymes
commercially available the most commonly used are the oxidases, such as glucose oxidase
(GOx) and horseradish peroxidase (HRP), beta-lactamase, urease, tyrosinase, and acetylcho
line esterase or choline oxidase.
The biocomplexing or bioaffinity sensors are based on antibodies, natural biological

chemoreceptors, nucleic acids, aptamers or molecularly imprinted polymers (MIPs). These
biosensors provide selective interactions of the analyte with a given ligand to form a thermody
namically stable complex. The analyte interacts with macromolecules or organized molecular
assemblies that have either been isolated from their original biological environment or
http://dx.doi.org/10.5772/60510
engineered. Thus, equilibrium is usually reached and there is no further consumption of the
analyte by the immobilized biocomplexing agent. These equilibrium responses are either
monitored by the integrated detector or by using a complementary biocatalytic reaction. The
most developed examples of biosensors using biocomplexing receptors are based on immuno
chemical reactions, i.e., the binding of an antigen (Ag) to a specific antibody (Ab), and will be
discussed in detail in the chapter concerning immunosensors.
The biological chemoreceptors [18] are used for drug and neurotransmitter detection, and they
are based on an affinity reaction. Using nucleic acids as a biocomponent, one can detect the
interaction between the synthetic drug and the genetic material and also the hybridization
processes by working with single-strand DNA [19, 20].
The use of microorganisms and of whole vegetal or animal tissues is complex and very often their
selectivity is limited [21].
3. Nanomaterials involved in biosensor construction
Advances in nanotechnology have led to the recent development of many nanomaterials,

including carbon nanomaterials, magnetic and metallic nanoparticles [22]. Nanomaterials,
generally defined as materials with feature sizes smaller than 100 nm, have a remarkable
impact on various fields of application due to their properties (enhanced electrical conductiv
ity, tensile strength and chemical reactivity), which are imparted by their increased surface
area per unit weight. Nanomaterials have already been applied in electronics, foods, cosmetics,
electronics, drug development and sensing devices. The use of nanomaterials, especially
nanoparticles and nanostructured films, offers advantageous properties that can be exploited
in order to maximize the interactions with specific bioelements, to maintain their activity,
minimize structural changes, and enhance the catalytic step. In the biosensor field, the
analytical exploitation of such protein-nanomaterial interactions are an emerging trend that
spans many disciplines [23]. Nanomaterials based on metals, semiconductors and organic
compounds contribute to the enhancement of optical, electrical, chemical and magnetic
properties that are relevant in the case of sensing devices, and due to these facts they have been
frequently used in this research field [24, 25].
Nanomaterials are mainly used for electrode construction or modification and as biomolecule
tracers. Nanoparticles (NPs) are very stable (compared to enzyme labels), offering high
sensitivity (thousands of atoms can be released from one nanoparticle) and a wide variety are
available on the market. Nanoparticles are used nowadays as electrochemical labels or as
vehicles containing several hundreds or thousands of electroactive labels, pushing detection
limits down to several hundreds of biomolecules [26].
Various nanomaterials have been successfully applied for the immobilization of bioelements,
such as metallic nanoparticles, carbon or metallic nanotubes, nanosilver-coated magnetic
beads, magnetic particles, functionalized conductive polymers, etc.
3.1. Carbon-based materials
Different types of carbon nanostructures are becoming more and more popular due to their
specific structures, properties and the possibility of being used for many applications. In fact,
a wide variety of carbon-based materials are available, such as nanoparticles, nanodiamonds,
nano-onions, peapods, nanofibers, nanorings, fullerenes and nanotubes, which have been
extensively used in analytical applications. The basic structure of fullerenes and nanotubes
consists of a layer of sp2-bonded carbon atoms. This configuration, which resembles that of
graphene, is responsible for their good electrical conductivity and their ability to form charge-
transfer complexes when in contact with electron donor groups [27]. This configuration is
responsible for the development of strong van der Waals forces that significantly hamper the
solubility and dispersion of carbon-based nanoparticles. In order to avoid these problems,
different pretreatment methods have been proposed [28, 29, 30], such as the addition of polar
groups (oxygen, hydroxyl, phenyl and polyvinylpyrrolidone). The surface defects could also
affect the stability, the mechanical and electrical properties of carbon nanostructures [31, 32,
33, 34].
The carbon nanomaterials (Figure 1) cover a broad range of structures, beginning with zero-
dimensional structures (fullerenes, diamond clusters), continuing with one-dimensional
(nanotubes), two-dimensional (graphene), and three-dimensional structures (nanocrystalline
diamond, fullerite).
Diamond Graphite Amorphous

carbon
Fullerene C60 Fullerene C540 Carbon onions
Graphene CNT: SWCNT CNT: MWCNT
Figure 1. Main carbon entities at nanoscale level
Carbon nanomaterials, including fullerenes, graphene and carbon nanotubes, have many
technological advantages such as facile modification by functional groups, high carrier
http://dx.doi.org/10.5772/60510
capacity, incorporating both hydrophilic and hydrophobic substances, and high chemical
stability.
3.1.1. Carbon nanotubes
Within the family of nanomaterials, carbon nanotubes (CNTs), described for the first time by
Iijima [35], are arousing growing interest in relation to several modern devices, such as
transducers, for the improvement of biosensing systems, mainly due to their unique structural,
electronic and chemical properties, which include large surface area, high electrical conduc
tivity, and excellent properties of electron transfer reactions. Furthermore, CNTs have a unique
tubular structure, good biocompatibility and modifiable sidewall, making them ideal candi
dates for the construction of sensors with high performances [36].
Electrochemical nanobiosensors have been increasingly used in various types of nanomate

rials, mainly CNTs, as electrochemical transducers, in order to improve them from the
perspectives of automation, miniaturization and multiplexed analysis, but also to im
prove the analytical performances of such devices [37, 38]. For example, the detection of
diseases at an early stage is one of the goals in developing and improving CNT biosen
sors, focusing on their sensitivity, fast response and small sample volume, in order to
provide new strategies for the detection of specific biomarkers at low concentrations in
complex sample media (e.g., serum) [39].
Many properties and the quality of CNTs are directly influenced by the way in which the
graphene sheets are wrapped around [40], and they are affected by the operating conditions
in their fabrication process. The production methods generally require transition metal
nanoparticles (i.e., Fe, Co and Ni) to function as growth catalysts. When the catalysts are in
contact with a gaseous carbon or a hydrocarbon source, the resulting carbon is deposited on
the particle surface and the CNT grows rapidly at the surface of the catalyst. The diameter of
the CNTs, their quantity and quality can be controlled by varying some reaction parameters
(e.g., temperature, metal concentration, gas type and pressure). The diameter of the NPs
defines the nature of the CNTs, whether single-walled (SWCNTs) with diameters in the range
0.4-3 nm, or a group of concentric CNTs sharing a common axis constituting double- and
multiwalled (MWCNTs) with diameters in the range 1.4-100 nm [36].
In the case of CNT structures, the reactivity of atoms situated in the plane is different from
those at the edges. For a number of biologically important compounds, the electrochemis
try of atoms located at the edge-plane sites of the CNT is comparable to that of different
planes of graphite [41], and the metal and non-metal impurities contained determine some
of the electrochemical catalytic properties and limitations of CNTs [42, 43, 44]. It is also
worth mentioning that the electrode surface modification with CNTs leads to a changed
mass transport regime from semi-infinite diffusion to effective thin-layer diffusion in a
porous layer, and the increased current may frequently be erroneously assigned to
electrocatalysis [45, 46, 47].
Currently, SWCNTs and MWCNTs are synthesized by high-temperature processes (e.g.,

electric arc discharge and laser ablation, which are physical methods to convert carbon into
NTs) and chemical vapour deposition (CVD), which is performed at lower temperatures and
atmospheric pressure.
Usually, the nanostructured materials obtained using CNTs were morphologically charac
terized using transmission electron microscopy (TEM) and scanning electron microscopy
(SEM). Figure 2 presents the SEM images obtained for SWCNT [48] and MWCNT [49].
A B
Figure 2. (A) SEM image of SWCNT composite film [48]; (B) FE-SEM images of MWCNT in high magnification [49]
Many papers published in recent years report CNT biosensors based on different oxidases
applied for the detection of glucose [50, 51, 52, 53, 54], H2O2 [55, 56, 57], L-lactate [58, 59, 60],
acetaminophen [61, 62, 63, 64, 65], etc. The construction and optimization of new biosensors
with HRP immobilized onto the transducer surface, either screen-printed electrode (SPE) or
glassy carbon electrode (GCE), by entrapment into polymer films (polyethylenimine (PEI) and
polypyrrole (Ppy)) doped with SWCNT and MWCNT, was described [61, 66].
The obtained configurations were used to monitor the signal produced by the electrochemical
reduction of the enzymatically generated electroactive N-acetylbenzoquinoneimine (the
oxidized form of acetaminophen) in the presence of hydrogen peroxide using amperometry,
cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in synthetic and real
samples, with the best limit of detection (LOD) obtained for acetaminophen of 1.36 M [61].
3.1.2. Graphene
Graphene is another material that offers great prospects for the future of analytical chemistry,
consisting of a one-atom-thick sheet of sp2 hybridized carbon atom composed of six member
rings that provide a surface area that is nearly twice as large as that of SWCNT [43]. This
material presents high mechanical strength, high elasticity and high thermal conductivity [43],
but also presents a major disadvantage: its poor dispersion in aqueous medium. Stable
dispersions of graphene sheets have been achieved using amphiphilic polymers, alkylamines
and hydrophilic carboxyl groups, among others, as dispersing agents [67]. The functionaliza
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tion of graphene is considered an important route for improving its dispersion. Therefore,
different authors have tried producing graphene oxide, a graphite derivative with hydroxyl,
epoxy and carboxyl groups covalently attached to its layers, with a better dispersion in some
solvents being obtained.
Graphene can be interlinked with CNT for the fabrication of high-performance transparent
electrodes, and the resulting films present features comparable to indium tin oxide [68]. The
electrochemical activity of crystalline graphene is different from that of reduced graphene
oxide flakes [69]. A wide variety of methods are available for graphene fabrication, the first
being published in 2004 [70], and consist in the mechanical exfoliation (repeated peeling) of
small patches of highly ordered pyrolytic graphite. Many other fabrication methods have been
developed, including unzipping MWCNT to form graphene ribbons [71, 72], substrate-
independent methods using micromoulding inside a capillary [73], and spray deposition of
graphene oxide-hydrazine dispersions [74].
SEM images of a graphene film deposited on GCE revealed the typical crumpled and wrinkled
graphene sheet structure on the rough surface of the film (Figure 3 A). The TEM image
presented in Figure 3B shows the wrinkled graphene sheet with no aggregation, indicating
that the functionalized graphene sheets were well dispersed in ethanol, forming stable
suspensions [75].
A B
Figure 3. (A) SEM image of graphene-film-modified GCE. (B) TEM image of graphene in ethanol [75]
With regard to aptasensor development, graphene looks like it could have the necessary
requirements to implement next-generation and high-performance aptasensors. Graphene
naturally adsorbs the unfolded aptamer through the stacking interaction between the
purine and pyrimidine bases and the graphene plane, and hinders the adsorption of the folded
aptamer [76, 77].
The currently developed strategies for graphene functionalization with aptamers include
covalent and non-covalent approaches. The covalent approach often relies on the oxidation or
the reduction of graphene oxide that presents carbonyl and carboxyl functional groups [78].
The density of carbonyl and carboxyl functional groups can be increased through chemical
oxidation, which is widely used for the chemical modification of the inert graphitic structure
[79]. Because this procedure affects the properties of graphene, the covalent modification of
graphitic surface through a free radical addition reaction is used, without a preoxidation step
and with less impact on the electronic properties [80]. Besides the immobilization methods
mentioned above, amide [81] and phosphoramidate reactions [82] were employed towards the
fabrication of graphene-based biosensors. Some graphene derivatives, such as graphene oxide,
were also used as transducers in electrochemical biosensor development [83].
Graphene can also mediate electron transfer in view of the high-density edge-plane-like defects
present within its nanostructure. In the absence of a target, aptamers immobilized on the gold
electrode adsorb graphene nanosheets due to the strong interaction that accelerates the
electron transfer ratio between the electroactive species and the electrode surface. In the
presence of a target, the binding reaction inhibits the adsorption of graphene and blocks the
electron transfer. A label-free electrochemical aptasensor was constructed by taking advantage
of the ultra-fast electron transfer ratio of graphene [84]. This detection strategy was further
improved by nuclease for interferon gamma, yielding an LOD of 0.065 pM [85]. Other authors
[86] employed graphene oxide nanoplatelets (GONPs) as electroactive labels for thrombin
detection.
A simple label-free electrochemical immunosensor was constructed by modifying a graphite-
based SPE with graphene oxide after functionalization with N-hydroxysuccinimide in the
presence of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride, Figure 4.
Figure 4. Steps involved in the antiacetaminophen Ab-based immunosensor development; GO=Graphene oxide;
NHS=N-hydroxysuccinimide; EDAC=1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride; Ab anti
APAP=antibody antiacetaminophen; APAP=acetaminophen; SWV=square wave voltammetry [87]
The above-described immunosensor was applied with good results for the determination of
acetaminophen in synthetic and real samples by using square wave voltammetry (SWV) with
an LOD of 0.17 M [87].
3.1.3. Carbon nanoparticle-based electrochemical sensor

A sensitive and selective electrochemical sensor fabricated via the drop-casting of a suspension
of carbon nanoparticles (CNPs) onto a GCE was investigated in simultaneous determination
of acetaminophen and tramadol in pharmaceuticals and human plasma. CV and DPV studies
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of acetaminophen and tramadol were carried out at the modified and bare GCE. The results
of the electrochemical investigations showed that CNPs enhanced the electroactive surface
area and caused a remarkable increase in the peak currents, due to the diffusion within the
porous layer. Enhanced sensitivity and a considerable decrease in the anodic overpotential
leading to negative shifts in peak potentials was obtained with the modified GCE. By using
DPV, the sensor showed good sensitivity and selectivity: LODs for acetaminophen and
tramadol were 0.05 and 1 M, respectively [88].
3.2. Inorganic and organic nanoparticles
Nanoparticles of major environmental, pharmaceutical and biomedical importance include

magnetic nanoparticles, quantum dots, metal nanoparticles, silica nanoparticles and polymeric
types with intrinsic properties contributing to their use in specific applications. Typically,
nanoparticles are classified into: magnetic nanoparticles (possessing a core of Fe3O4) frequently
used in drug delivery and lately in immunosensing; semiconductor/QDs (based on CdS, CdSe)
used in bio- and immunosensing; metal nanoparticles (Ag, Au, Pd) used in biosensing and drug
delivery; polymeric nanoparticles (polystyrene) mainly used in drug and gene delivery; hybrid
nanoparticles (carbon or Fe3O4 covered with different materials like metal oxides, polymers,
amino acids, etc.) [89].
3.2.1. Magnetic nanoparticles
In recent years, considerable efforts were made to develop magnetic nanoparticles (MNPs),
due to their inherent advantages (magnetism, nanosize) and low cost of production. MNPs
with 10-20 nm diameters exhibit their best performance, due to supermagnetism, which makes
them especially suitable for a fast response. Due to their inherent dimensions, they possess a
large surface area and high mass transfer capacity [90].
MNPs can be integrated into the transducer materials and/or be dispersed in the sample
followed by their attraction by an external magnetic field onto the active detection surface of
the (bio)sensor. Biorecognition molecules like enzymes, antibodies or oligonucleotides
immobilized onto magnetic particles can be easily trapped by magnets and retained close to
or on an electrode surface [90, 91]. Since the properties of MNPs depend strongly on their
dimensions, their synthesis and preparation have to be designed in order to obtain particles
with adequate size-dependent physicochemical properties. MNPs possessing adequate
physicochemistry and tailored surface properties have been synthesized under precise
conditions and applied for sensors, biosensors and other detection systems [92, 93].
Magnetic particles consist of a magnetic core surrounded by a non-magnetic shell. Iron oxides,
such as magnetite (Fe3O4) or maghemite (-Fe2O3), are often used as core materials instead of
iron due to their higher stability [94]. For biological applications, magnetic particles generally
consist of nanosized superparamagnetic iron oxide distributed through a matrix, mainly
composed of silica [95] or an organic polymer [96]. Silica-based magnetic beads can be prepared
in different shapes and sizes with different degrees of porosity, which are much more attractive
for the immobilization of biomolecules due to their chemical and mechanical stability and their
resistance to bacterial attack [97]. Many types of magnetic particles, functionalized with
different groups like carboxyl, amino, hydroxyl or epoxy, are currently available for different
applications.
The majority of MNP-based systems use inorganic nanocrystals as magnetic cores. The
composition of these inorganic nanocrystal ranges from metals and alloys to metal oxides.
Among the metal oxide compounds, superparamagnetic iron oxide nanoparticles, including
magnetite (Fe3O4) and maghemite (-Fe2O3), are the most popular [98]. The surfaces of the
particles can be modified with organic polymer or inorganic compounds (e.g., gold, silica,
alumina), suitable for further functionalization by the attachment of various bioactive mole
cules [99]. The functionalization of MNPs with bioelements exhibits specificity, due to both
the bioreceptor and the magnetism.
MNPs amplify the electrochemical signal, improving the sensitivity of electrochemical devices
[90], through their contact with the electrode surface, transport of a redox-active species to the
electrode surface, and formation of a thin film on the electrode surface. The detection for MNP-
based electrochemical biosensors can be performed by potentiometry [100, 101], amperometry
[102, 103], electrochemiluminescence [104, 105], electrochemical impedance spectroscopy [106,
107] and especially voltammetry [108, 109, 110, 111, 112, 113, 114, 115].
Protein G-MNP-based label-free electrochemical immunosensor was elaborated for the
ultrasensitive and specific detection of acetaminophen using a carbon-based SPE as immobi
lization platform (Figure 5). An LOD of 1.76 M was calculated based on the calibration data.
The performances of the optimized immunoassay were tested using two pharmaceutical
products containing acetaminophen with excellent recoveries [116].
Figure 5. Protein G-MNP-based label-free electrochemical immunosensor development [116]
Protein G-functionalized magnetic beads and graphite-based SPEs were also used to develop
an HRP-labelled sandwich type immunosensor for Mucin 1 (MUC1) electrochemical detection
with LOD of 1.34 ppb [117].
Fe3O4 magnetic dipolar attraction and its large surface area/volume ratio may lead to aggre
gation in clusters when exposed to biological solutions. Functionalization can overcome this
problem and also enhance biocompatibility. Many strategies were used for the functionaliza
tion of MNPs, such as core-shell Au-Fe3O4 [109], core-shell Au-Fe3O4@SiO2 [102], core-shell
Fe3O4@SiO2 [112], Au-Fe3O4composite NPs [108], Fe3O4@SiO2/MWCNTs [103], Fe3O4 anchored
on reduced graphene oxide [113] and Fe3O4@ Au-MWCNT-chitosan [114]. Among all these,
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core-shell Fe3O4@SiO2 contributes to the stabilization of MNPs in solution and enhances the
binding of ligands at the surface of MNPs. Electrode surface modified with core-shell
Fe3O4@SiO2 shows good electrical conductivity and more electroactive interaction sites,
providing enhanced mass transport and easier accessibility to the active sites, thus increasing
the analytical signal and sensitivity [90]. A comparison between the analytical performances
of some recently developed electrochemical sensors and biosensors based on MNPs is
presented in Table 1.
Detection method MNPs type Analyte LOD Reference
Voltammetry Au-Fe3O4 Organochloride pesticides 56 pg/mL [108]
Voltammetry Core-shell Au-Fe3O4 Carcinoembryonic Ag 10 ng/mL [109]
Voltammetry Fe3O4 Au NP Clenbuterol 220 ng/mL [110]
Voltammetry Fe3O4 Au NP H2O2 20 M [111]
Voltammetry Core-shell Fe3O4@SiO2 Metronidazole 18 nM [112]
Voltammetry Fe3O4-RGO Cr(III) - [113]
Fe3O4@Au-MWCNT-
Voltammetry Streptomycin 1.5 nM [114]
chitosan
Core-shell Fe3O4@SiO2/
Voltammetry Uric acid 0.13 M [115]
MWCNT
Potentiometry MaMB Dynabeads Protein G Zearalenone 7 ng/mL [100]
Potentiometry Core-shell-Fe3O4 Glucose 0.5 M [101]
Amperommetry Core-shell Au-Fe3O4 @SiO2 Glucose 0.01 mM [102]
Amperommetry Fe3O4@SiO2-MWCNT Glucose 800 nM [118]
EIS Fe3O4-COOH/MNP OchratoxinA 10 pg/mL [106]
Fe@Au NP-2-
EIS aminoethanethiol DNA 2 fM [107]
- graphene NP
Detection method MNPs type Analyte LOD Reference
Table 1. Selected examples of sensors and biosensors based on magnetic nanoparticles
3.2.2. Metal nanoparticles
Metal nanoparticles and CNTs, have been employed to immobilize biomolecules for the
construction of sol-gel biosensors, due to their large surface area, high catalytic activity and
good biocompatibility. Moreover, the excellent conductivity of gold nanoparticles (AuNPs)
provides the potential to construct amperometric biosensors based on redox enzymes (as
sensing elements) and nanoparticle arrays (as conductive matrices where enzyme molecules
are immobilized) [118, 119]. Metal nanoparticles, such as Pd and Pt, were deposited on
SWCNTs and MWCNTs in order to increase the electrocatalytic activity of CNTs. An improved
conductivity and electrocatalytic behaviour was obtained by combining sol-gel materials with
CNTs, the electron transfer between the redox enzyme and the electrode being facilitated [120].
Gold nanomaterials have unusual optical and electronic properties, high stability and
biological compatibility. Aptamer-conjugated gold nanomaterials provide powerful
platforms to facilitate targeted recognition, detection and therapy applications, due to their
controllable morphology, size dispersion and easy surface functionalization [121].
Many applications of nanoparticles, such as metal NPs (gold, silver) or semiconductor dots in
biosensing, have been recently reported [122, 123]. A DNA array detection system based on
oligonucleotide targets labelled with AuNPs was developed [124], and AuNPs or silver-
enhanced colloidal gold were used as labels for electrochemical detection of DNA hybridiza
tion [125, 126, 127]. These NP-based electrochemical biosensors employed the stripping
voltammetric technique as the read-out principle due to the preconcentration step resulting in
ultra-trace-level LODs.
AuNPs are irreversibly immobilized on the transducer, the electrode surface being difficult to
regenerate. To overcome this problem, Chen et al. [128] have developed a novel electrochemical
system for the sensitive detection of glucose (LOD of 1 mM) as well as single-nucleotide
polymorphism, which has the AuNPs directly in the supporting electrolyte. This one-pot
detection system can be operated and regenerated very easily, since all the components are
integrated in the electrolyte of the gold colloid and the unmodified electrode can be reused.
The sensing mechanism of potentiometric biosensors using NPs is based on a sandwich assay
(Figure 6), the target being captured by the immobilized primary receptor and then attached
to the NP-labelled secondary receptor. Potentiometry is finally used to detect the dissolution
products of NPs after a release step with H2O2 [123].
Miniaturized ion-selective electrodes (ISEs) designed to detect ions in microvolume samples,
with a concentration of fM, were reported [129, 130], and an LOD of 12.5 pmol/50L was
obtained for IgG using an ISE based on silver-enhanced AuNPs [131]. Metal NPs can also be
used as a catalyst for the reduction of metal ion, via the enzymatically generated reducing
agent, and the biometallization processes can be monitored by ISEs in real time [132]. Thereby,
an ISE based on AuNPs was developed and applied for highly sensitive detection of glucose
[132, 133], while the NADH potentiometric detection was successfully obtained by monitoring
the NADH-stimulated catalytic reduction of Cu2+ in the presence of AuNPs (which have a
crucial role in improving the sensitivity) with a Cu2+-ISE [134]. Wang et al. [135] reported a
highly sensitive potentiometric biosensor for detecting DNA hybridization based on the
depletion of Ag+ ions induced by the biocatalytic reaction of alkaline phosphatase, recorded
in the presence of AuNPs. The sensor has been used to detect DNA and 16S rRNA of Escherichia
coli with LOD of 50 fM, using only 4 L of sample solution.
Two simple electrochemical aptasensors were developed for the detection of Mucin 1 (MUC1)
tumour marker based on its specific recognition by thiolated aptamers immobilized on Au
NP-modified graphite and gold SPEs. The quantitative detection of MUC1 protein was
electrochemically achieved by electrochemical impedance spectroscopy (EIS) and DPV. The
http://dx.doi.org/10.5772/60510
Figure 6. Potentiometric biosensors using nanoparticles as labels [123]
estimated detection limits for MUC1 were 3.6 ng/mL at AuNP modified graphite SPE by EIS
and 0.95 ng/mL at AuNP modified gold SPE by DPV [136].
3.2.3. Nanosized clays

Nanosized clays can serve as matrices for electroactive species because they are usually able
to incorporate ions by an ion exchange process. Moreover, adsorption of proteins on clay
mineral surfaces plays a very important role in the development of biosensors. By modifying
the electrode surface with clays, the heterogeneous electron transfer process between the
protein and the electrode surface is facilitated.
The entrapment of biomolecules in the clay matrix represents an inexpensive, fast and easy
method for the elaboration of enzyme electrodes. The procedure consisting in the adsorption
of an enzyme/clay aqueous colloid mixture onto the electrode surface offered the possibility
of entrapping a large and known amount of enzyme into the clay film, and could also be
adapted for interdigitated microelectrodes [137].
Mesoporous silica nanoparticles can be easily synthesized by using surfactant templating
methods [138]. Silica nanoparticles have become an important platform in biomedical appli
cations and bioanalysis because of their high density, which facilitates separation through
centrifugation during their synthesis, and their modification and detection steps [139]. The
silica surface also provides a biocompatible and versatile substrate for biomolecule immobi
lization, required for further utilization in biosystems and for drug delivery [140]. Following
typical DNA silica surface conjugate chemistry, the aptamer immobilization on silica nano
particles has been well developed for various applications [141].
Biosensors based on clay materials can play an important role as efficient tools in the field of
forensic analysis considering their well-known advantages of sensitivity, selectivity, simple
functioning and affordability [142]. Thereby, a bienzyme biosensor based on acid phosphatase
(AcP) and polyphenol oxidase (PPO) simultaneously entrapped in anionic clays was con
structed and applied for the specific amperometric determination of As(V). The biosensor
relied on the successive hydrolysis of phenyl phosphate to phenol catalysed by AcP and the
oxidation of phenol to o-quinone catalysed by PPO. The electrochemical reduction of the
formed o-quinone was accomplished at a detection potential of -0.2 V vs Ag/AgCl with a
detection limit of 0.15 ng/mL [143]. DNA-functionalized, layer-by-layer assembled SWCNT
hybrids with clay nanoparticles were proposed for the detection of As(III) achieving an LOD
of 0.05 mg L-1 [144]. Another simple amperometric biosensor for cyanide involved the immo
bilization of PPO into ion exchanger clay based on Zn-Al double-layered hydroxides (LOD of
0.1 nM). The low LOD was a consequence of the anion accumulation in the ion exchanger clay
which also contained the immobilized enzyme [145].
3.2.4. Nanosized and nanostructured polymeric films
One of the most important aspects of biosensor development is the bioelement immobilization
platform, which needs to ensure high stability and maintain its activity as long as possible.
Several types of conductive or insulating polymers were used to obtain a nano/micropat
terned surface with applications in the biosensor field [146]. Electropolymerization in the
presence of a template, removed after the polymer formation, is an efficient route for the
synthesis of 2D and 3D conductive polymers (CPs). Among the conductive polymers, poly
pyrrole and polyaniline [147, 148] were extensively studied because of their electrochemical
properties, aqueous compatibility and their ability to form nanostructures like nanowires,
nanotubes or microcontainers [149].
Hydrogels are cross-linked hydrophilic polymer structures that can hold large amounts of
water or biological fluids in their pores. As one of the newest classes of polymer-based systems,
target-responsive hydrogels have found numerous biomedical and pharmaceutical applica
tions [121, 150]. Polyacrylamide/acrylate is a copolymer of acrylamide and acrylic acid, and
acrydite-modified oligonucleotides can form hydrogels by the introduction of proper com
plementary DNA as a cross-linker. He et al. [151] reported an aptamer-based reversible DNA
induced hydrogel system for molecular recognition and separation.
Different types of hydrogel nanoparticles were used together with various biocomponents,
such as enzymes, proteins and nucleic acids, during the development of electrochemical
biosensing systems, in order to increase their sensing performances [152].
CPs and in particular functionalized CPs are useful for biosensor construction as a platform
to immobilize the primary Ab on the electrode surface. Poly o-aminobenzoic acid (PABA) can
be considered a carboxyl-functionalized aniline CP, capable of self-doping, and is also a soluble
derivative of polyaniline. Its carboxyl group can be exploited as a linker, either for PABA
immobilization onto the electrode, or for covalent bond formation with biomolecules such as
proteins and antibodies [153].
http://dx.doi.org/10.5772/60510
An electrochemical aptamer-based biosensing assay for MUC1 protein detection by the

modification of the graphite-based SPEs surface using a functionalized conductive polymer
(PABA), and by using methylene blue (MB) as an electrochemical indicator was developed.
The recognition of immunoreactions and aptamer binding events was assessed by monitoring
the interfacial electron transfer resistance with EIS, while CV and DPV were employed for
sensitive indirect quantification of MUC1 via MB with a detection limit of 0.62 ppb [154]. In
order to establish the optimal configuration of HRP-based biosensors able to detect acetami
nophen in various matrices, another conductive polymer, polypyrrole, was deposited on GCE
and SPE by several methods. Chronoamperometric studies performed proved the interaction
between the immobilized enzyme and the electroactive species of paracetamol, NAPQI,
enzymatically generated in the presence of H2O2 [155].
The combination of nanosphere lithography, electrodeposition and click chemistry was

applied for the immobilization of ferrocene at GCE. The nanostructuration of the surface
was obtained by adsorption of 100 and 900 nm diameter polystyrene nanospheres fol
lowed either by electropolymerization of N-(10-azidodecyl) pyrrole or by electrografting of
4-azidobenzenediazonium. This approach offered potential applications such as engineer
ing of porous materials and was used for HRP immobilization. The nanostructured
biosensor was applied for acetaminophen detection in the presence of hydrogen perox
ide, and showed better performances in terms of sensitivity and LOD in comparison with
a non-structured electrode [156]. Another template based on nanostructured polymeric film
was developed combining latex nanosphere lithography with electropolymerization of N-
substituted pyrrole monomer in order to immobilize both tyrosinase and GOx and to obtain
a biosensor with higher sensing performances [148].
3.2.5. Molecularly imprinted polymers
MIPs can be successfully used in applications relying on selective molecular binding as they
are fully artificial macromolecular structures with biomimetic properties, imitating receptor-
ligand, Ab-Ag, or enzyme-substrate biorecognition [157].
MIPs possess several advantages over their biological counterparts, including low cost, ease
of preparation, long term stability and shelf life, inherent reusability, high mechanical strength,
and remarkable chemical and thermal stability under various experimental conditions,
allowing the very specific binding of the analyte in harsh chemical media [158, 159].
The rational design of MIPs is still considered a challenge due to the numerous experimental
variables to be addressed, and it has been previously reviewed in detail by several groups of
authors [157, 158, 160, 161] along with their most important analytical applications.
MIPs prepared by bulk polymerization have proven to be poorly compatible with transducers
in terms of efficient immobilization strategies, mass transfer and rebinding kinetics, even if
they are ground before use. Therefore, several aspects related to the in-situ polymerization,
and more specifically to the electrosynthesis of MIPs and hybrid technologies involving MIPs
in the fabrication of biosensors, as well as their most recent advances in addressing two
important challenges for MIP-based electrochemical biosensors, the determination of bioma

cromolecules and enantiospecific analysis, will be very briefly presented.
Electroactive organic functional monomers can polymerize to form conducting, semiconduct

ing or insulating polymers. Polymerization may be performed chemically (using an oxidizer
as external reagent) or electrochemically (by electropolymerization). Electropolymerization is
the more convenient approach, in terms of simplicity and time-effectiveness, for electrochem
ical biosensor fabrication as it leads to a tight binding of an MIP film onto the surface of the
transducer. This procedure ensures both the synthesis and the surface immobilization of the
biomimetic layer, along with the possibility of controlling polymer nucleation and growth,
film thickness and morphology by the proper selection of the electropolymerization parame
ters. The reproducible preparation of thin MIP layers is of the utmost importance in sensing
applications, because it reduces response time by significantly shortening diffusional path
lengths. Solvent swelling and the inclusion of the supporting electrolytes ions tunes the
rigidity and porosity of the MIP film. Electropolymerization may be performed under
galvanostatic, potentiometric or most frequently potentiodynamic conditions, in both non-
aqueous and aqueous solutions. The first two techniques are able to generate well-ordered,
charged polymeric layers doped with counter ions for charge neutralization, whereas the latter
generates a more tangled, charged or neutral polymeric matrix due to the continuous influx
and release of solvated ions during its growth upon film charging and discharging [157].
Using electroinsulating MIP films in the fabrication of electrochemical sensors carries a major
disadvantage due to the high resistance to charge transfer and the lack of direct path for
electron conduction from the recognition sites to the electrode transducer, suffering from an
incomplete template removal upon their overoxidation. Using electronically conducting
polymers for MIP synthesis avoids these undesirable effects.
Some of the most common electroinsulating layers employed in MIP technology are acrylic-
and vinylic-based polymers, but the most frequently reported electrosynthesized insulating
MIPs are polyphenylenediamine, polyphenol, polyaminophenol and polythiophenol. Due to
their low conductivity, sensors based on such MIPs usually use optical, piezoelectric or
impedimetric signal transduction. However, as long as they are electrodeposited in a thickness
that does not insulate the underlying conductive electrode surface (<10 nm) [162], a successful
voltamperometric signal transduction may also be performed [163].
In the case of electrosynthesis of conducting MIP layers for biosensing, electroactive

functional monomers such as pyrrole, thiophene, aniline, 3-aminophenylboronic acid and
porphyrin derivatives are most often reported. Analyte determination using such conduct
ing layers may be performed by any available signal transduction procedure, such as
optical, piezoelectric, voltammetric, potentiometric, chronoamperometric, impedimetric or
conductometric procedures. Poly(pyrrole), poly(thiophene), poly(aniline) and their deriva
tives are amongst the most widely studied conducting polymers [157, 160], due to their
high conductivities in their oxidized form and their ability to reversibly switch between
conducting and insulating states by doping and undoping [160]. As mentioned earlier, in
the non-covalent molecular imprinting, a wide variety of templates are amenable to be used
with elevated imprinting efficiencies, ranging from small analytes to macromolecules
http://dx.doi.org/10.5772/60510
(proteins, nucleotides). Additional promising approaches include implementing surface MIP

for cells (yeast), bacteria, virus or blood [164, 165, 166]. In the case of using small organic
molecules as templates, MIP synthesis is well established, however the imprinting of much
larger molecules is still considered to be a challenge. Larger templates are less rigid, which
does not ensure the formation of well-defined binding cavities during macromolecular
imprinting, and, furthermore, the secondary and tertiary structures of biomacromolecules
(i.e., proteins) may be also affected under the imprinting conditions. The large size hinders
the protein both in reaching and leaving the artificial binding sites, and their molecular
complexity increases the chances of non-specific binding with the MIP, leading to poor
selectivity and cross-reactivity. More importantly, proteins are often incompatible with the
organic solvents used in MIPs synthesis. The use of an aqueous solution for macromolecu
lar imprinting greatly restricts the choice of reactive functional monomers and cross-
linkers. Additionally, water could compete for and potentially disrupt any hydrogen bonds
between the template and the functional monomers. The protein as template in macromo
lecular imprinting may be introduced by three different approaches: it may be dissolved
into the prepolymerization mixture (bulk imprinting), grafted to the surface of the
transducer followed by a partial inclusion into the bulk polymer with the resulting binding
sites situated on the MIP surface (surface imprinting), or only a small epitope part of the
protein (epitope imprinting) may be used as a template, resulting in an MIP that is able to
recognize the whole target protein. Surface and epitope imprinting enhance the imprint
ing effect for biomolecules, leading to higher affinity and capacity for the template, as well
as to reduced non-specific binding; they improve protein binding kinetics, overcoming
difficulties to mass transfer and protein removal in imprinted matrices. While surface
imprinting is an efficient technique, the conditions used to accomplish the imprinting
process must be carefully considered. The conformational stability of proteins is sensitive
to their surrounding conditions, including temperature, pH, ion concentration, surfactant
and its concentration, the ratio of template to functional monomers, the types of function
al monomers used, and initiator type [167]. MIPs generated by epitope imprinting mani
fest minimal non-specific binding and improved affinity. Furthermore, it is less costly
working with short peptides as templates, and more importantly, because of the tem
plates higher stability, organic solvents may also be used during the imprinting process.
The detection of the bound non-electroactive target macromolecule is performed indirect
ly, in the presence or absence of a redox probe, by various signal transduction principles,
such as voltammetry, piezomicrogravimetry, impedance spectroscopy, etc.
The high selectivity of such biomimetic polymers is also demonstrated by their efficient use
for the molecular imprinting of optically active templates. This special application of MIP-
based chiral electrochemical sensing has not been fully exploited, even though there is a great
necessity in the pharmaceutical industry for developing fast and cost-effective methods of
chiral analysis applicable from the early stages of drug development. The vast majority of MIP-
based electrochemical sensors were developed for non-chiral analysis. Only around 20 articles
report MIP-based sensors designed for the chiral analysis of different small-molecules, but not
pharmaceutically active compounds, like amino acids [168, 169, 170] or monosaccharides [171].
Moreover, enantiospecific signal transduction was only reported in the case of amino acids.
Nevertheless, very recently an MIP-based electrochemical sensor has been reported for the
simultaneous enantiospecific recognition of several -blocker enantiomers [163].
In spite of the electroinsulating properties of the R(+)-atenolol imprinted acrylate-based MIP,

voltamperometric signal transduction was successfully performed, due to the resulting very
thin (less than 4 nm) and porous MIP layer. The sensor exhibited distinctive enantiospecific
oxidation peaks towards the R-antipodes of four -blocker representatives and additional
oxidation peaks common to both enantiomers of each studied -blocker, thus allowing the
simultaneous analysis of all of their enantiomers in a single determination.
3.3. Hybrid materials
Metal NPs decorating CNTs or graphene sheets, carbon-coated magnetic NPs, nanoparticles
included in carbon paste electrodes, MWCNT-alumina-coated silica, and insulating MIPs
combined with various nanostructures are examples of some hybrid nanomaterials used for
the enhancement of biosensor sensitivity.
3.3.1. CNT-based hybrid materials
Electrodes modified by hybrid materials, consisting of CNTs and metal nanoparticles, have
been developed for use as fuel cell catalysts and biosensors [172]. For example, platinum
nanoparticles (PtNPs) catalyse the electrochemical oxidation of H2O2, which is generated by
the enzymatic reaction, while MWCNTs can be employed to modify the working electrode for
the enlargement of the electroactive surface area [173]. A highly sensitive Pt-CNT-glucose
biosensor was developed using the incorporation of GOx on a Pt-CNT electrode but with poor
enzyme stability after storage [174]. Electrodes modified by CNTs/PtNPs hybrids protected
by dendrimers were first developed for methanol electrochemical oxidation [175], and then
adapted for the detection of inorganic phosphorous pesticide through the inhibition of the
acetylcholinesterase [172]. These electrochemical biosensors are based on a hybrid material,
consisting of MWCNTs, PtNPs and poly(amidoamine) dendrimer (DEN) as a binder for
enzyme immobilization (Figure 7).
Figure 7. Biosensor preparation by loading MWCNT, PtNPs and enzyme through dendrimer binders [172]
MWCNTs increase the surface area and promote the electron transfer properties, PtNPs
intensify the electron transferability in addition to catalytic performance, and dendrimers play
a role in the uptake of the redox agent as well as the binder functionality.
http://dx.doi.org/10.5772/60510
Two new xanthine biosensors, based on GC modified with chitosan (CS), Co3O4NPs and
MWCNTs were developed [176]. Two different enzyme systems were investigated involving
monoenzyme xanthine oxidase (XO) immobilization and bienzyme xanthine oxidase
(XO)/HRP co-immobilization. Xanthine amperometric determination was achieved by both
electrooxidation and electroreduction of the enzymatically generated hydrogen peroxide, with
LODs of 0.2 M and 20 M with anodic and cathodic processes, respectively. The proposed
biosensors were applied for detecting xanthine in fish with average recoveries of 96.23.5 %
with XO/Co3O4MWCNTs/CS/GCE and 95.42.1 % with XO/HRP/Co3O4MWCNTs/CS/GCE,
respectively.
3.3.2. Graphene-based hybrid materials

Graphene can be decorated with AuNPs to enhance the signal transduction or to increase the
grafting area for biocomponent immobilization. AuNPs are the most commonly used metal
nanoparticles in graphene-based biosensing systems, mainly due to their ability to form strong
covalent bonds with the thiol groups [26].
An electrochemical aptasensor using graphene-AuNPs composite obtained by the reduction
of tetrachloroauric acid with sodium citrate in a graphene water suspension was reported
[109]. Direct electrodeposition technique of AuNPs was also applied to graphene-based
electrochemical transducers yielding a high density of nanoparticles that provide an ultra-
large specific surface area available for aptamer immobilization [177].
A graphene-CNTs-incorporating zinc oxide NPs hybrid composite (GR-CNT-ZnO) was
synthesized and applied to fabricate an enzyme-based glucose biosensor (Figure 8).
Figure 8. The preparation of GR-CNT-ZnO composite [178]
By using CV, authors have concluded that the modified film in the presence of oxygen has
high electrocatalytic ability in glucose detection with a wide linear detection range and a
LOD of 4.5 M of glucose. The sensor was successfully applied for human serum sam
ples, and the results were in good agreement with those determined using a standard
photometric method [178].
3.3.3. Molecularly imprinted polymer-based hybrid materials
Hybrid materials combining insulating MIPs with electronically conducting polymers tend to
correct the drawbacks of the former and feature networks of molecular wires connecting
recognitions sites to the electrode surface [179]. Molecularly imprinted nanoparticles, nano
spheres, nanoshells or nanofibres obtained by various synthesis procedures (precipitation or
emulsion/suspension polymerization, mini/emulsion precipitation, etc.), along with various
organic/inorganic hybrid materials [165, 180, 181], metal nanoparticles, such as Ag [182] and
Au [183] or carbon nanomaterials [173], may be combined with electrosynthesized MIP films,
leading to composite MIP/nanomaterial biosensing systems [160, 161] with larger specific
surface area, higher sensibility and specificity, and higher biocompatibility for trace electro
chemical analysis of small analytes and biomacromolecules.
4. Perspectives and future development
The selection of materials and fabrication techniques is crucial for adequate sensor function
and the performance of a biosensor often ultimately depends upon these, rather than upon
other factors. Consequently, future developments in biosensor design will inevitably focus
upon the technology of new materials, especially the new copolymers that promise to solve
the biocompatibility problem and offer the prospect of more widespread use of biosen
sors in clinical monitoring. The future trends in sensor development that are expected to
have an impact on biosensor performances mainly concern immobilization techniques,
nanotechnology, miniaturization and design of multisensor arrays.
Author details
Robert Sndulescu*, Mihaela Terti, Cecilia Cristea and Ede Bodoki
*Address all correspondence to: rsandulescu@umfcluj.ro
Analytical Chemistry Department, Iuliu Haieganu University of Medicine and Pharmacy

Cluj-Napoca, Romania
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New Materials For The Construction of Electrochemical Bosensors

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Selection of our books indexed in the

Chapter from the book Biosensors - Micro and Nanoscale Applications

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New Materials for the Construction of Electrochemical

Robert Sndulescu, Mihaela Terti,

Additional information is available at the end of the chapter

Keywords: Carbon-based nanomaterials, metal nanoparticles, magnetic nanoparticles, nanostruc

materials and nanostructured architectures (i.e., nanoparticles, carbon nanotubes and

2. Strategies of electrochemical biosensor technology

The biocatalytic recognition sensors are based on a reaction catalysed by macromolecules,

The biocomplexing or bioaffinity sensors are based on antibodies, natural biological

3. Nanomaterials involved in biosensor construction

Advances in nanotechnology have led to the recent development of many nanomaterials,

3.1. Carbon-based materials

Diamond Graphite Amorphous

Fullerene C60 Fullerene C540 Carbon onions

Graphene CNT: SWCNT CNT: MWCNT

Figure 1. Main carbon entities at nanoscale level

3.1.1. Carbon nanotubes

Electrochemical nanobiosensors have been increasingly used in various types of nanomate

Currently, SWCNTs and MWCNTs are synthesized by high-temperature processes (e.g.,

3.1.3. Carbon nanoparticle-based electrochemical sensor

3.2. Inorganic and organic nanoparticles

Nanoparticles of major environmental, pharmaceutical and biomedical importance include

3.2.1. Magnetic nanoparticles

Figure 5. Protein G-MNP-based label-free electrochemical immunosensor development [116]

Detection method MNPs type Analyte LOD Reference

Voltammetry Au-Fe3O4 Organochloride pesticides 56 pg/mL [108]

Voltammetry Core-shell Au-Fe3O4 Carcinoembryonic Ag 10 ng/mL [109]

Voltammetry Fe3O4 Au NP Clenbuterol 220 ng/mL [110]

Voltammetry Fe3O4 Au NP H2O2 20 M [111]

Voltammetry Core-shell Fe3O4@SiO2 Metronidazole 18 nM [112]

Voltammetry Fe3O4-RGO Cr(III) - [113]

Potentiometry MaMB Dynabeads Protein G Zearalenone 7 ng/mL [100]

Potentiometry Core-shell-Fe3O4 Glucose 0.5 M [101]

Amperommetry Core-shell Au-Fe3O4 @SiO2 Glucose 0.01 mM [102]

Amperommetry Fe3O4@SiO2-MWCNT Glucose 800 nM [118]

EIS Fe3O4-COOH/MNP OchratoxinA 10 pg/mL [106]

Detection method MNPs type Analyte LOD Reference

Table 1. Selected examples of sensors and biosensors based on magnetic nanoparticles

3.2.2. Metal nanoparticles

Figure 6. Potentiometric biosensors using nanoparticles as labels [123]

3.2.3. Nanosized clays

3.2.4. Nanosized and nanostructured polymeric films

An electrochemical aptamer-based biosensing assay for MUC1 protein detection by the

The combination of nanosphere lithography, electrodeposition and click chemistry was

3.2.5. Molecularly imprinted polymers

important challenges for MIP-based electrochemical biosensors, the determination of bioma

Electroactive organic functional monomers can polymerize to form conducting, semiconduct

In the case of electrosynthesis of conducting MIP layers for biosensing, electroactive

(proteins, nucleotides). Additional promising approaches include implementing surface MIP

In spite of the electroinsulating properties of the R(+)-atenolol imprinted acrylate-based MIP,

3.3. Hybrid materials

3.3.1. CNT-based hybrid materials

3.3.2. Graphene-based hybrid materials

Figure 8. The preparation of GR-CNT-ZnO composite [178]

3.3.3. Molecularly imprinted polymer-based hybrid materials

4. Perspectives and future development

Robert Sndulescu*, Mihaela Terti, Cecilia Cristea and Ede Bodoki

*Address all correspondence to: rsandulescu@umfcluj.ro

Analytical Chemistry Department, Iuliu Haieganu University of Medicine and Pharmacy